Serine protease isoforms of Deinagkistrodon acutus venom: cloning, sequencing and phylogenetic analysis

The major coagulating fibrinogenase of Deinagkistrdon acutus venom. designa ted acutobin, was purified by anion-exchange chromatography, gel filtration and reverse-phase HPLC. Approximately 80% of its protein sequence was dete rmined by sequencing the various fragments derived from CNBr cleavage and d igestion with endoprotease. Extensive screening of the venom gland cDNA spe cies after amplification by PCR resulted in the isolation of four distinct cDNA clones encoding acutobin and three other serine proteases, designated Dav-PA, Dav-KN and Dav-X, The complete amino acid sequences of these enzyme s were deduced from the cDNA sequences. The amino-acid sequence of acutobin contains a single chain of 236 residues including four potential N-glycosy lation sites. The purified acutobin (40 kDa) contains approx. 30% carbohydr ate by weight: which could be partly removed by N-glycanase. The phylogenet ic tree of the complete amino acid sequences of 40 serine proteases from 18 species of Crotalinae shows functional clusters reflecting parallel evolut ion of the three major venom enzyme subtypes: coagulating enzymes, kininoge nases and plasminogen activators. The possible structural elements responsi ble for the functional specificity of each subtype are discussed.